RSPO3 is a novel contraction-inducible factor identified in an “in vitro exercise model” using primary human myotubes

The physiological significance of skeletal muscle as a secretory organ is now well known but we can only speculate as to the existence of as-yet-unidentified myokines, especially those upregulated in response to muscle contractile activity. We first attempted to establish an “insert-chamber based in vitro exercise model” allowing the miniature but high cell-density culture state enabling highly developed contractile human myotubes to be readily obtained by applying electric pulse stimulation (EPS). By employing this in vitro exercise model, we identified R-spondin 3 (RSPO3) as a novel contraction-inducible myokine produced by cultured human myotubes. Contraction-dependent muscular RSPO3 mRNA upregulation was confirmed in skeletal muscles of mice subjected to sciatic nerve mediated in situ contraction as well as those of mice after 2 h of running. Pharmacological in vitro experiments demonstrated a relatively high concentration of metformin (millimolar range) to suppress the contraction-inducible mRNA upregulation of human myokines including RSPO3, interleukin (IL)-6, IL-8 and CXCL1. Our data also suggest human RSPO3 to be a paracrine factor that may positively participate in the myogenesis processes of myoblasts and satellite cells. Thus, the “insert chamber-based in vitro exercise model” is a potentially valuable research tool for investigating contraction-inducible biological responses of human myotubes usually exhibiting poorer contractility development even in the setting of EPS treatment.

In vitro exercise models using contractile cultured myotubes, with electric pulse stimulation (EPS) 1 application, have been widely employed for elucidating the muscle cell responses induced by actual contractile activity, including their myokine expressions 2,3 . Unlike in vivo skeletal muscles, in vitro exercise models contain only myotubes with no other cell types and thus allow solely the contraction-inducible biological responses of the myotubes to be evaluated 4,5 . This major advantage is especially valuable for investigating human myotubes since, for a variety of reasons, human exercise experiments are obviously difficult to perform as compared to animal exercise experiments. However, human myotubes derived from primary satellite cells obtained from human subjects usually exhibit much poorer contractility development than those of murine myotubes such as those originating from C2C12 myoblasts even with EPS treatment 6,7 . While we recently established a "feeder-supported in vitro exercise model" by co-culturing with murine fibroblast cells that markedly facilitated the contractility development of human myotubes 8 , a useful and simple method allowing human myotubes alone, with no escorting cells, to be readily adapted to an in vitro exercise model is eagerly anticipated.
We herein succeeded in establishing a novel "in vitro exercise model" applicable to human-derived muscle cells, which is based on utilization of a specialized insert chamber that efficiently delivers EPS to myotubes grown Cell culture. Primary human satellite cells were obtained by fluorescent-activated cell sorting (FACS) from muscle biopsy tissues at Tohoku University Hospital under the approval of the Ethics Committee of Tohoku University, and written informed consent was obtained from the patient (see below). The primary human satellite cells were amplified and stocked in liquid N 2 as previously reported 9 . Primary satellite cells were cultured in a growth medium containing DMEM/Ham's F10 mixture supplemented with 20% FBS, 1% penicillin-streptomycin, 1% chicken embryonic extract (United States Biological, Salem, MA, USA), and 2.5-ng/ml basic fibroblast growth factor (Thermo Fischer Scientific, Waltham, MA, USA) at 37 °C under a 5% CO2 atmosphere.
Human myoblasts (activated satellite cells) were seeded at a density of 6.6 × 10 4 cells in the insert chamber placed on a 24-well plate in 1 mL of growth medium. We used the insert (culture area = 0.47 cm 2 , the size of which corresponds to a well of the 24-well plate) and seeded human myoblasts in a high-cell-density state (6.6 ~ × 10 4 cells/0.47 cm 2 /insert), resulting in an approximately 12-fold higher cell density than those in routine in vitro exercise models using an 8-well culture plate (1.25 × 10 5 cells/10.5 cm 2 /well) 7 . For immunofluorescent analysis, human myoblasts were seeded onto a Cell-Disc LF in a 24-well plate at a density of ~ 1 × 10 5 cells. Two days after plating, differentiation was induced by switching the growth medium to DMEM supplemented with 5% horse serum, 30 µg/ml penicillin, and 100 µg/ml streptomycin (differentiation medium). The differentiation medium was changed every 24-48 h during the 7-8 days of differentiation.
In some experiments, C2C12 cells were also used for evaluating the insert-chamber-based in vitro exercise model (Fig. S1). C2C12 myoblasts were seeded at a density of 1 × 10 4 cells in the insert chamber placed on a 24-well plate in 1 mL of DMEM supplemented with 20% FBS. Two days after seeding, differentiation was induced by switching to differentiation medium 1 and the cells were then subjected to the EPS experiments. The differentiation medium was changed every 24-48 h during the 7-8 days of differentiation.
siRNA-mediated reduction of RSPO3. Silencer select siRNAs against human RSPO3 (s39542, s39543), scramble siRNAs (#4,390,844, #4,390,847) and Lipofectamine RNAi MAX transfection reagent were purchased from Thermo Fischer Scientific (Grand Island, NY, USA). For myogenesis experiments, siRNAs were transfected by Lipofectamine RNAi MAX at day 0 of differentiation and then continuously cultured for 6-7 additional days with differentiation medium. For myotube experiments, siRNAs were transfected at day 5 of differentiation and cultured for 2 additional days with differentiation medium, and the cells were then used for the EPS experiments.
Electrical pulse stimulation (EPS). After 7-8 days of differentiation, the insert chamber and the Cell-Disc were transferred to an 8-well plate (two chambers or two Cell-Discs per well) placed in a C-Dish (IonOptix, Milton, MA) for EPS. EPS (1 Hz, 1 ~ 6 ms, 20 V/25 mm) was applied to the cells in the C-Dish using a C-Pace 100 pulse generator (IonOptix). DMEM containing 2% CS supplemented with final two-fold concentrations of amino acids, achieved by adding 50X amino acid solution (Thermo Fisher, #11130036) and 100X non-essential amino acid solution (Thermo Fisher, #11140050), was used during the EPS treatments, as previously reported 1 . A total of 4, 8, 16 or 24 h of EPS was applied and the cells were then harvested for analyses. In the experiments with a total of 16 or 24 h of EPS, 1 h intervals were set between the 8 h EPS sessions to prevent unwanted detachment of vigorously contracting myotubes from the substratum of the insert-chamber 7 .
Western blot analysis. The cells were harvested in lysis buffer (50 mM Tris-HCl, pH7.4, 150 mM NaCl, 20 mM sodium pyrophosphate, 10 mM NaF, 2 mM sodium orthovanadate, 1 mM EDTA, 1% Triton X-100, 1 μg/ml pepstatin, 5 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 6500 IU/ml aprotinin, phosphatase inhibitor mixture-1, Sigma), and after end-over-end rotation of the homogenates for 30 min, lysate supernatants were collected by centrifugation (15,000 rpm) for 20 min at 4°C, and protein concentrations were measured using the bicinchoninic acid method with bovine serum albumin (BSA) as the standard (Pierce). After electro-  www.nature.com/scientificreports/ conjugated anti-CD56 (clone MEM-188), and PE-conjugated anti-PDGFRα (clone 16A1). The negative set included blood markers CD11b and CD45, and endothelial markers CD31 and CD34. While CD34 is known to be expressed by the majority of mouse satellite cells 10 , human muscle-derived CD34 + cells are myogenic and adipogenic, whereas CD34 − cells are myogenic but not adipogenic 11 . We therefore used CD34 as a negative selection marker. Human satellite cells were defined as single live mononuclear CD11b − CD31 − CD34 − CD45 − CD56 + cells. FACS was performed on a FACS ARIA II flow cytometer (BD Biosciences). Cells were cultured in a growth medium and cultured at 37 °C in a 5% CO 2 atmosphere. When cells reached 60%-80% confluence, adherent cells were split onto a new Matrigel-coated 15-cm dish to expand the activated satellite cells. Activated satellite cells (myoblasts) were suspended in Cell Banker (TAKARA, CB011, Japan) and stored in liquid nitrogen.
In Situ Muscle Contraction and Running Exercise. Male 7-9-week-old Balb/c mice were purchased from CLEA-Japan (Tokyo, Japan). The experimental design, as well as the care and use of the mice, followed the guidelines for animal experiments of Tohoku University and the Ethics Committee for Animal Experiments, Tohoku University, approved these studies (Ethics approval #2020BeLMO-006-01). The study was carried out in compliance with the ARRIVE guidelines and the aforementioned guidelines. The mice were kept in standard cages in an air-conditioned room under a 12:12 light/dark cycle at 23 ± 1°C. They were allowed access to standard food pellets (24.2% protein, 4.5% lipids, 56.8% carbohydrates) (Labo MR Standard, Nihon Nosan Kogyo Co., Yokohama, Japan) and tap water ad libitum. Mice that had been fasted for 16 h were anesthetized with an intraperitoneal injection of medetomidine (ZENOAQ, 0.3 mg/kg), midazolam (SANDZ, 4.0 mg/kg), and butorphanol (Meiji Seika Pharm CO, 5.0 mg/kg). The sciatic nerves were bilaterally isolated, and subminiature electrodes were placed around each nerve and interfaced with the electrical stimulation instrument (STG4004, Multi Channel Systems, Reutlingen, Germany) as previously reported 12 . Hindlimb muscles on one side were stimulated to induce contractions with 1 s on/1 s off cycles for 3 min (2.5 mA amplitude, 50 Hz, 1 ms duration), and other leg served as a sham-operated control. At 1 h after EPS, SOL and EDL muscles were isolated from the mice and then subjected to RT-PCR analysis. Our EPS setting was determined by monitoring upregulations of CXCL1 and IL-6 mRNAs in SOL and EDL muscles ( Fig. S3), confirming that these two factors are well-established as exerciseinducible myokines in both in vivo 1,5 and in vitro 4,7,13 experimental systems. Forced wheel running was imposed on mice at room temperature (23 ± 1 °C) for the indicated time in each experiment, as previously described but with slight modifications 14 . Briefly, each mouse was placed in a cylindrical cage (17 cm diameter, 7 cm width) with stainless steel rods (2 mm diameter, 1 cm apart), which was rotated by an electric motor at a walking speed of ~ 12 m/min. In total, 12 running wheels were operating simultaneously. The SOL and EDL muscles were isolated from the mice immediately after 2 h of running and then subjected to RT-PCR analysis.
Statistical analysis. Statistical analyses were performed using Student's t-test or ANOVA with Tukey's multiple comparison test, and P values < 0.05 were considered to indicate a statistically significant difference. Data are expressed as means ± SE unless otherwise specified.
Ethics declarations. The experimental design, care, and use of the mice were performed according to the guidelines for animal experiments at Tohoku University. Ethical approval for this study was obtained from the Animal Research Committee of Tohoku University (approval number; #2020BeLMO-006-01).

Results
Effect of EPS on RSPO3 mRNA expression in human myotubes cultured in the insert chamber. Given that a high-cell-density inoculation of human-origin myoblasts (activated satellite cells) improves their contractility development along with better upregulation of myokines in response to EPS 7 , we used the insert chamber allowing us to maintain the culture in a high-cell-density state (6.6 ~ × 10 4 cells/culture), resulting in better contraction-inducible biological responses of human myotubes with EPS treatment. As shown in Figs. 1A-C, we confirmed that human myotubes in the insert chamber were highly responsive to EPS treatment as assessed by upregulations of the expressions of contraction-inducible mRNAs including those of myokines (CXCL1 and IL8) 1,7 and STARS 15 . Since EPS (1 Hz, 20 V/25 mm) with a 2 ~ 4 ms pulse duration produced maximum responses of all mRNA upregulations examined (Figs. 1 and S1), we hereafter applied EPS with a 4 ms pulse duration for all experiments.
Preliminary RNA-seq analysis identified a significant increase in RSPO3 mRNA expression in response to EPS-induced contractile activity when using the in vitro exercise model (DDBJ repository, Accession number: PRJDB12901). We performed RT-PCR analysis for RSPO1-4 mRNA expression levels and confirmed the EPSinduced RSPO3 upregulation in human myotubes cultured in the insert chamber to occur in a manner dependent on the pulse duration of EPS (Fig. 1D). In contrast, no expressions of RSPO1 and RSPO4 mRNAs were detected, and the RSPO2 mRNA expression level did not change in response to EPS in human myotubes (Fig. S2).
Western blotting analysis of whole cell lysates from human myotubes demonstrated that EPS significantly increased the RSPO3 protein amounts broadly observed around 35 ~ 40 kDa ( Fig. 2A), due perhaps to its N-glycosylation 16 and C-mannosylation 17 . We observed approximately 37 kDa bands even in the control myotubes, but their nature and relationships to other EPS-inducible bands broadly detected around 35 ~ 40 kDa are unclear. Densitometric evaluation of all bands, including that at 37 kDa, demonstrated significant increases in RSPO3 protein amounts after EPS treatment (Fig. 2B). The EPS-dependent increase in RSPO3 protein was further confirmed by immunofluorescent staining analysis using anti-RSPO3 antibody (Fig. 2C). expression during differentiation of human myoblasts, we performed RT-PCR analysis and found that RSPO3 expression was slightly increased upon myogenic differentiation. In fully differentiated myotubes (day 8), RSPO3 expression was markedly upregulated by EPS treatment (Fig. 3A). Contraction-inducible RSPO3 upregulation diminished to ~ 50% at 4 h after stopping EPS and returned to the basal level the next day, indicating that RSPO3 mRNA upregulation is entirely dependent on contractile activity elicited by EPS treatment in differentiated myotubes under the culture conditions employed in this study (Fig. 3B). Given that several MAP-kinase signaling cascades appear to be involved in EPS-dependent myokine expressions in myotubes 13 , pharmacological inhibitors were used to examine whether EPS-dependent RSPO3 mRNA upregulation was impacted by chemical reagents (Fig. 3C). EPS-induced RSPO3 upregulation was enhanced in the presence of SP-600129 (JNK inhibitor), or PD-98056 (MEK1/2 inhibitor), whereas SB-203580 (p38 MPAK inhibitor) slightly but significantly inhibited RSPO3 upregulation (Fig. 3C), implying that these MAP kinase cascades play intricate roles in the observed contraction responsiveness.
Effects of siRNA-mediated silencing of RSPO3 on EPS-inducible myokine upregulation and myogenic differentiation. To examine the possible involvement of RSPO3 in the processes by which other myokines are upregulated in response to EPS-evoked contraction, siRNA-mediated RSPO3 knockdown experiments were carried out. In this set of experiments, siRNAs were transfected into myotubes 2 days before EPS treatment. Under conditions wherein RSPO3 expression was significantly suppressed by specific, but not scramble, siRNA pretreatment, no obvious impact of RSPO3 silencing was detected and EPS-dependent upregulations of IL-6, IL-8 and CXCL1 were similar to those observed in the control groups (Fig. 4). These results suggest that RSPO3 is not directly involved in EPS-inducible upregulation of several other myokines, at least not IL-6, IL-8 and CXCL1, in differentiated myotubes.
The involvement of RSPOs, particularly RSPO1 and 2, in myogenic differentiation and fusion processes has previously been reported 18,19 . We therefore examined effects of siRNA-mediated silencing of RSPO3 in myoblasts on myotubular formation (Fig. 5A) as assessed by sarcomeric α-actinin immunofluorescent staining analysis and   6D) were found to be more sensitive to metformin and, in fact, a lower concentration (1.25 mM) of metformin significantly inhibited the EPS-dependent response. Western blot analysis confirmed that metformin markedly stimulated phosphorylation of both AMPK and ACC (Fig. 6F). These results suggest EPS-dependent mRNA expression of several myokines including RSPO3 to be negatively affected by the presence of metformin.

Effects of sciatic-nerve-mediated muscle contraction and running exercise on RSPO3 expression in soleus and EDL muscles.
To examine contraction-dependent muscular RSPO3 upregulation in vivo, we utilized an in situ mouse muscle contraction model with sciatic nerved-mediated electrical pulse stimulation (SN-EPS). RSPO3 mRNA levels increased significantly in both EDL and SOL muscles in response to SN-EPS treatment as compared with those in sham control legs (Fig. 7A). As positive controls, increased expressions of IL-6 and CXCL1 mRNAs were confirmed in EDL and SOL muscles which had undergone SN-EPS-mediated contractile activity ( Figure S3). Wheel running experiments (2 h) demonstrated RSPO3 mRNA levels to be significantly increased in SOL muscles. Although EDL muscle showed a tendency for RSPO3 mRNA upregulation in response to 2 h of running, the increase did not reach statistical significance (Fig. 7B).

Discussion
While "in vitro exercise models", employing mostly murine muscle cells such as C2C12 cells, have been widely used for investigating contraction-inducible biological responses including myokine expressions 1,13,24 , humanderived myotubes have generally exhibited much poorer contractility development, resulting in minimal myokine expressions even with EPS treatments 6,7 . In the present study, we established an "insert chamber-based in vitro exercise model" applicable to human myotubes that allows highly-developed EPS-responsive human myotubes to be readily obtained. By using the insert possessing electric conductive path on the wall, myotubes on the PET membrane in the insert could be effectively exposed to EPS and thus exhibited myokine upregulations sensitively in a manner dependent on EPS intensity (e.g., pulse duration) (Fig. S1). EPS-inducible biological responses in the insert chambers exhibited an EPS-intensity dependence, being maximal at a 2 ~ 4 ms EPS pulse duration, essentially the same responsiveness as that observed in the conventional culture plate 1,4,7,8 . Therefore, miniaturization along with higher-cell-density using the insert not only contributes to better development of human myotube contractility but also allows for marked increases in sample numbers, which would be an important advantage especially for primary human myotubes derived from biopsy samples, a very limited resource. By employing our "insert chamber-based in vitro exercise model comprised of primary human myotubes", we focused on the effects of EPS on human RSPO3 mRNA expression since exercise-dependent regulation of RSPO3 expression had yet to be investigated, despite its physiological significance in various muscular functions      www.nature.com/scientificreports/ having been revealed in experimental animal studies 18,19,[25][26][27][28][29] .The findings presented herein demonstrate that mRNA expression of RSPO3, but not other RSOPs, in human myotubes is highly sensitive to EPS treatment (Figs. 1 and S2), features very similar to those of other established contraction-inducible myokines including IL-6, IL-8, and CXCL1 1,4,13 . Western blotting and immunofluorescent staining analyses confirmed that human myotubes did indeed produce RSPO3 protein in response to EPS treatment (Fig. 2). While these results clearly indicated RSPO3 to be produced by human myotubes especially in response to EPS treatment, we were not able to measure RSPO3 proteins secreted into the culture medium by the ELISA method, due to detection limits of the assay. We used a small insert (culture area = 0.47 cm 2 ) to optimize cell density and then placed two of these inserts in a well of an 8-well plate containing a relatively large volume of culture medium (5 mL/10.5 cm 2 /well). Therefore, we can reasonably assume that a small amount of RSPO3 secreted into the medium would be extremely diluted and thus be present at a concentration below the detection limit of the assay. Interestingly, as we reported previously for EPS-dependent IL-6 upregulation in murine C2C12 myotubes 4 , EPS-induced RSPO3 mRNA upregulation was relatively delayed and a significant increment was observed after 16 h of EPS treatment (Fig. 3A). Importantly, EPS-induced RSPO3 mRNA upregulation was restored to the basal level after cessation of EPS (Fig. 3B), further illustrating RSPO3 expression to be predominantly regulated by the EPS-dependent contractile activity in myotubes, possibly serving as an exercise factor in vivo. Consistent with this notion, we observed RSPO3 mRNA in mouse skeletal muscles to be significantly upregulated in both SOL and EDL muscles, subjected to SN-EPS-evoked in situ contraction, while no such elevation was detected in those of sham control legs (Fig. 7). Exercise-inducible upregulation of muscular RSPO3 was further confirmed in the SOL muscles after 2 h of running, while EDL muscles did not exhibit significant RSPO3 upregulation (Fig. 7B). Although it is quite difficult to directly compare these in vitro results with data obtained in vivo, one of the explanations for the discrepancy in the time differences required for RSPO3 expression between in vitro and in vivo is that acquisition of RSPO3 upregulation capability in myotubes may represent the maturation process of contraction-dependent responsiveness, as is similarly observed with IL-6 that reportedly required more than 12 h of EPS 4 . Taking these in vitro and in vivo data together, our findings provide compelling evidence that www.nature.com/scientificreports/ RSPO3 expression is regulated by muscle contractile activity and also suggest that RSPO3 serves as a contractioninducible myokine in vivo.
RSPOs are secretory proteins that play important roles in cell proliferation, differentiation and death by evoking the Wnt signaling pathways in various cell types 30 . In the skeletal muscle research field, physiological roles of RSPOs, especially RSPO1-3, have been well documented in myogenesis 18,25 , muscle development 26 , muscle repair/ regeneration 19,27 , and acetylcholine receptor clustering 28,29 in animal models including mice and xenopus, though RSPO studies in human muscle cells have been lacking. Thus, based on our present findings, future human studies are warranted to determine whether and how muscular RSPO3 expression is stimulated by physical activity and/or contractile activity of skeletal muscles in vivo.
Pharmacological and siRNA-mediated silencing experiments conducted in the present study provide important insights into regulatory mechanism(s) involving contraction-dependent RSPO3 mRNA upregulation as well as into its possible physiological roles in muscle cells. As is evident from Fig. 3C, multiple MAP kinase cascades play intricate roles in RSPO3 expression in both positive and negative fashions in human myotubes. Unlike the contraction-dependent CXCL1 upregulation that reportedly relies mainly on the JNK MAPK pathway 13 , SP600129, a JNK inhibitor, slightly but significantly augmented RSPO3 upregulation, a phenomenon which was also detected in contraction-induced IL-6 upregulation 4 . PD98056, a MEK1/2 inhibitor, also exerted a positive effect on RSPO3 upregulation, while SB20358, a p38 inhibitor, slightly but significantly suppressed this response. Thus, intracellular signaling pathways involved in the contraction-dependent upregulation of each myokine apparently differ from each other, and the EPS-induced RSPO3 upregulation is likely to be balanced by the multiple MAP kinase cascades that may contribute to deciphering the complicated signals evoked by the actual contractile activity of myotubes. Clarifying intracellular signaling networks involved in the contraction-dependent myokine upregulations is fundamental for our understanding of exercise physiology 31 , and this important issue needs to be addressed by future experiments.
An interesting observation made in the pharmacological experiments depicted in Fig. 6 was that a relatively high concentration of metformin (~5 mM), the first-line drug for type 2 diabetes 32,33 , slightly but significantly suppressed the upregulation of all contraction-inducible myokines examined in the present study including RSPO3 (Fig. 6). Intriguingly, EPS-induced expressions of RSPO3 and STARS mRNAs were moderately more sensitive to this reagent and a lower concentration of metformin (1.25 mM) significantly suppressed their upregulations in response to EPS treatment. However, the millimolar range of metformin doses, representing supra-pharmacological concentrations, was required for the inhibitory effect on contraction-inducible responses including those of RSPO3 and STARS, suggesting mitochondrial respiratory chain complex I inhibition and thereby AMPK activation 34,35 to likely be involved in the suppressive effects. Indeed, AMPK phosphorylation was markedly augmented by metformin (2.5 mM) treatment in human myotubes (Fig. 6). Given that metformin reportedly dampened some beneficial adaptations induced by exercise training in several clinical trials 21,22,36 , our in vitro results suggest that the impact of metformin on the contraction-inducible expressions of myokines including RSPO3 may, by as yet unknown mechanisms, be related to the clinical effects of metformin on exercise benefits. Although we did not observe obvious cytotoxic effects of metformin as assessed by myotube morphology in the present study, a high concentration of metformin reportedly influenced cellular viability 37 and therefore caution must be exercised when interpreting the in vitro data particularly for supra-physiological concentrations of metformin. In any case, our "insert chamber-based in vitro exercise model" for human myotubes is a potentially valuable research tool for directly examining pharmacological actions on contracting human myotubes in greater detail, and future studies employing more sophisticated experimental conditions are necessary to understand the relationship between muscle functions, e.g., contractility, and metformin actions.
As mentioned above, the physiological roles of RSPOs, including RSPO3, in muscle functions have been intensively studied mostly by using experimental animals 18,19,[25][26][27][28][29] and their positive myogenic actions on myoblasts and satellite cells are well established. For example, RSPO2 reportedly enhanced the expression of MYF5, a key myogenic determination factor, mediated through WNT/β-catenin signaling, leading to hypertrophic myotube formation, and siRNA-mediated silencing of either RSPO2 or RSPO3 significantly compromised MYF5 expression in murine C2C12 myoblasts 18 . Consistent with the evidence obtained using animal models, we confirmed that siRNA-mediated RSPO3 silencing in human myoblasts inhibits their myotube formation (Fig. 5), implying the significance of RSPO3 for human muscular functions. Thus, our present findings not only confirmed the physiological significance of RSPO3 serving as a myogenic stimulator in human muscle cells but also strongly suggest that exercise-inducible RSPO3 serves as a local intramuscular factor which regulates myogenic cells neighboring the working myofibers that might be involved in hypertrophy and/or repair processes after exercise. Future studies are warranted to directly test whether RSPO3 functions as a myokine in regulating muscle functions by modulating myoblasts/satellite cells in vivo.
Given that siRNA-mediated silencing of RSPO3 in myotubes failed to exert any obvious effect on EPS-dependent upregulation of other myokines including IL-6, IL-8 and CXCL1 (Fig. 4), augmented RSPO3 expression is highly unlikely to be involved directly in the regulation of expressions of these myokines, at least as an autocrine factor. Taking these data together with those obtained in previous studies, the enhanced RSPO3 expression in response to contractile activity may serve as a positive myogenic factor for neighboring myoblasts/satellite cells, acting in a paracrine manner in contracting muscle tissues in vivo. Moreover, RSPO3 reportedly influenced, in animal studies, the body fat distribution via regulation of adipogenesis 38 , and hepatic cholesterol synthesis 39 . Thus, muscle derived RSPO3 enhanced by contractile activity might have an impact on these phenomena in adipose and/or liver tissues as an endocrine factor. In any case, further experiments are needed to define the physiological roles of muscle RSPO3 that is markedly enhanced by muscle cell contractile activity.
In summary, we herein provide compelling evidence that RSPO3 mRNA expression is highly upregulated in human myotubes by EPS treatment. These findings were obtained using a newly established "in vitro exercise model", which is based on the utilization of a specialized insert chamber. Our approach, using the insert chamber, Scientific Reports | (2022) 12:14291 | https://doi.org/10.1038/s41598-022-18190-z www.nature.com/scientificreports/ further expands the usefulness of the "in vitro exercise model", allowing us to maximize the use of human muscle cells obtained from clinical biopsy samples, a very limited resource.

Data availability
The RNA-seq datasets generated during the current study are available in the DDBJ repository, Accession No.: PRJDB12901. https:// ddbj. nig. ac. jp/ resou rce/ biopr oject/ PRJDB 12901. The data that support the findings of this study are available from the corresponding author, MK, upon reasonable request.